A unique antigen against SARS-CoV-2, Acinetobacter baumannii, and Pseudomonas aeruginosa

The recent outbreak of COVID-19 has increased hospital admissions, which could elevate the risk of nosocomial infections, such as A. baumannii and P. aeruginosa infections. Although effective vaccines have been developed against SARS-CoV-2, no approved treatment option is still available against antimicrobial-resistant strains of A. baumannii and P. aeruginosa. In the current study, an all-in-one antigen was designed based on an innovative, state-of-the-art strategy. In this regard, experimentally validated linear epitopes of spike protein (SARS-CoV-2), OmpA (A. baumannii), and OprF (P. aeruginosa) were selected to be harbored by mature OmpA as a scaffold. The selected epitopes were used to replace the loops and turns of the barrel domain in OmpA; OprF311–341 replaced the most similar sequence within the OmpA, and three validated epitopes of OmpA were retained intact. The obtained antigen encompasses five antigenic peptides of spike protein, which are involved in SARS-CoV-2 pathogenicity. One of these epitopes, viz. QTQTNSPRRARSV could trigger antibodies preventing super-antigenic characteristics of spike and alleviating probable autoimmune responses. The designed antigen could raise antibodies neutralizing emerging variants of SARS-CoV-2 since at least two epitopes are consensus. In conclusion, the designed antigen is expected to raise protective antibodies against SARS-CoV-2, A. baumannii, and P. aeruginosa.

Disrupting mutations. The disrupting property of mutations in the spike glycoprotein was evaluated at https:// weilab. math. msu. edu/ Mutat ionAn alyzer/. The database provides experimental data on the effect of mutations in weakening or abolishing the antigen-antibody affinity of known epitope-antibody complexes.
Antigen design. The OmpA of A. baumannii served as a scaffold to present the epitopes of spike glycoprotein and P. aeroginusa. To note, the experimentally validated OmpA epitopes remained intact. Therefore, the final construct would contain the collection of epitopes from the three mentioned pathogens.
The signal peptide and the last 15 aa of OmpA were removed based on the previous study 66 . The sequencebased strategy determined the best matching region of OmpA with OprF 311-341 . OmpA and OprF epitope (OprF 311-341 ) were aligned by ClustalW 103 at http:// www. ibi. vu. nl/ progr ams/ prali newww/. The matched region within OmpA was replaced with OprF 311-341 .
A structural approach was employed to find regions to replace by spike epitopes. Loops (except L3) and internal turns of OmpA β-barrel were replaced by the selected peptides of S 437-806 to present the epitopes of interest (Table 1).
The flexibility, surface accessibility, hydrophilicity, and beta-turn secondary structure of the designed constructs were assessed by available tools of IEDB. ProtParam server at https:// web. expasy. org/ protp aram/ was employed to estimate some physicochemical properties of the construct such as isoelectric point (pI) and instability index.
Beta-barrel OMP classification. The scaffold (OmpA) used for displaying epitopes is a known outer membrane protein. To predict whether this classification is retained after sequence replacements, the following servers were harnessed to predict mature OmpA and the construct classifications. The BOMP 108 (http:// servi ces. cbu. uib. no/ tools/ bomp/) is ranking integral OMPS in five ranks (1 to 5) in which 5 revealed the most reliable, and 1 indicates the least reliable prediction 108 . HHomp 109 (http:// toolk it. tuebi ngen. mpg. de/ hhomp) is employing an integrated beta-barrel prediction method to compare a generated profile of the Hidden Markov Model (HMM), from a query sequence, with a HMM database representing outer membrane proteins 109 . MCMBB 110 (http:// athina. biol. uoa. gr/ bioin forma tics/ mcmbb/) scores beta-barrel outer membrane proteins as > 0 (accuracy of > 90%) 110 .
Topology of the constructs. The topology of transmembrane proteins could be assisted in the accurate prediction of 3D structure. OMPs contain transmembrane β-strands, which could not be detected by transmembrane helix predictors. The topology of the designed construct was predicted by specialized transmembrane β-strand discriminators, viz. PRED-TMBB 111 at http:// bioin forma tics. biol. uoa. gr/ PRED-TMBB/ and BOCTOPUS2 112 at http:// bocto pus. cbr. su. se/ pred/ PRED-TMBB2 113 at http:// www. compg en. org/ tools/ PRED-TMBB2. PRED-TMBB is scoring a given sequence to predict whether the sequence is a beta-barrel outer membrane protein (< 2.965 are beta-barrel outer membrane). This tool provided three methods (Viterbi, N-best, and Posterior Decoding) to determine the topology of a given sequence. All the provided methods were employed for the prediction performance. The construct topology was compared to the OmpA topology.
Structure prediction and conformational epitopes. RaptorX-Property 114 at http:// rapto rx. uchic ago. edu/ Struc tureP roper tyPred/ predi ct/ was used to predict the secondary structure of the designed construct. RaptorX-Property predicts secondary structure, solvent accessibility, and disordered regions of a given protein sequence 114 . This server had been appointed as the best secondary structure predictor in an evaluation study 115 .
Several robust servers with different approaches were employed to predict the 3D structure of the designed construct. GalaxyWEB 116 at http:// galaxy. seokl ab. org/, FALCON@home 117 at http:// prote in. ict. ac. cn/ TreeTh read er/, I-TASSER 118 at http:// zhang lab. ccmb. med. umich. edu/I-TASSER/, Phyre2 119 at http:// www. sbg. bio. ic. ac. uk/ phyre2/ html/ page. cgi? id= index, ROBETTA 120 at http:// robet ta. baker lab. org/ and RaptorX 121 at http:// rapto rx. uchic ago. edu/. GalaxyTBM 122 in GalaxyWEB is a template-based modeler which builds the reliable core from multiple templates; then, it detects variable regions, such as loops, to be re-modeled by an ab initio method 122 . FALCON@home is a novel threading approach that uses remote homologous proteins as templates identified by an improved method 117 . I-TASSER uses multiple threading approaches to identify templates for iterative template-based fragment assembly simulation of full-length atomic models. This server had been ranked as the 1 st predictor in several community-wide CASP experiments 118 .
Phyre2 uses advanced methods to detect remote homologous for modeling a given protein. This server is ranked as a quarter 1 protein modeling tool in CASP9 and 10 119 . ROBETTA harnesses comparative modeling and de novo structure prediction methods to generate structural models 120 . RaptorX is a template-based protein structure modeling server appropriate for proteins with no close homolog in PDB 121 .
To screen the obtained models, the quality of the predicted structures was evaluated by QMEANDisCo 123 (https:// swiss model. expasy. org/ qmean/) and ERRAT 124 (https:// servi cesn. mbi. ucla. edu/ ERRAT/). QMEANDisCo is a single model method for the quality assessment of a predicted protein structure. This method combines statistical potentials and agreement terms with a distance constraint (DisCo) score indicating consistencies of pairwise CA-CA distances from a predicted structure with constraints of homologous structures 123 . ERRAT uses reliable high-resolution structures to show errors in a protein structure based on the statistics of non-bonded atom-atom interactions in the structure of interest 124 . Ramachandran plot of the models was delineated by PROCHECK 125 (https:// servi cesn. mbi. ucla. edu/ PROCH ECK/). Moreover, complying with the models and predicted secondary structure and topology of the construct were considered.
The best model was refined by 3Drefine 126 and GalaxyRefine 127 servers. 3Drefine refines the protein models by integrating iterative optimization of hydrogen bonds and energy minimization of the optimized model at the atomic level 126 . GalaxyRefine refines the models via rebuilding and repacking side chains followed by molecular dynamics simulation to perform overall structure relaxation. CASP10 assessment acknowledged GalaxyRefine as the most successful tool in improving the local structure quality 127 . The quality of refined models was evaluated by QMEANDisCo, ERRAT, and PROCHECK. An accurate reliable 3D structure is an essential input for the prediction of conformational B-cell epitopes in a given antigen. The best-refined model of designed all-inone antigen was submitted to ElliPro 128 at http:// tools. iedb. org/ ellip ro/. This tool predicts potential linear and conformational B-cell epitopes of a given protein structure. Moreover, DiscoTope server 129  www.nature.com/scientificreports/ method combines the calculation of contact numbers and a novel epitope propensity amino acid score of residues in spatial proximity 129 . The epitopes of interest were mapped on the best-refined 3D structure of the construct.

Recombinant expression improvement.
The protein sequence of the designed all-in-one antigen served as a query for the codon optimization tool of VectorBuilder at https:// en. vecto rbuil der. com/ tool/ codonoptim izati on. html to obtain optimized DNA sequence to be expressed in E. coli. The optimized DNA sequence was submitted to TIsigner (Translation Initiation coding region designer) 130 at https:// tisig ner. com/ tisig ner to optimize translation initiation sites. Opening energy (mRNA accessibility), which is specific to the expression hosts, is calculated and optimized. The expression score is predicted from the minimum to maximum level (0 to 100) for the input sequence and the optimized one.

B-cell epitopes of receptor binding motif (RBM), cleavage site, and fusion peptide regions.
To determine the location of B-cell epitopes of RBM, cleavage site, and fusion peptide, different algorithms were applied. Three out of four software tools have predicted epitopes within the mentioned motives of the spike glycoprotein (Supplementary Table S1). Data in Supplementary Table S1 suggest the overlap of prediction with experimentally confirmed epitopes of OprF and OmpA. The predicted epitopes in all scenes are not completely consistent with experimental results. For instance, LBtope failed to assign the experimentally validated epitopes of OmpA. In the case of OprF, true positive results were achieved. The selected epitopes of S 437-806 met at least one of the physicochemical properties appropriate for B-cell epitope predictions (Fig. 1). The average flexibility, hydrophilicity, and beta-turn secondary structure of S 437-806 were 0.997, 1.678, and 1.020 respectively.
All the selected peptides matched to experimentally validated B-cell epitopes of S SARS-CoV-2 glycoprotein. Moreover, QTQTNSPRRARSV and IYKTPPIKDFGGF shared similarities with human peptides (the data derived from BLAST search against IEDB epitopes; see "Methods").

The selected epitopes of SARS-CoV-2 could be recognized in important variants.
To precisely select the most species-inclusive epitopes the conservancy assay was done using the alignment approach. The epitopes of interest were compared to more than 81,000 submitted sequences of spike glycoprotein available at GISAID. The conservancy of epitopes was presented as sequence logos (Fig. 2). The sequence logos suggest the presence of minor variability within the selected epitopes (Supplementary Table S2). To evaluate the effect of mutations that disrupt or weaken the antibody-antigen interaction, all positions of epitopes were compared www.nature.com/scientificreports/ to available data. Although some disrupting mutations are observed in different variants, at least one epitope remains intact (compare Table 2 and Supplementary Table S2). Therefore, the vaccine-escape property of the virus is expected to rule out to a large extent. Moreover, a special comparison of the selected epitopes with sequences of important variants revealed a conservancy of epitopes. The conservancy of the 5 selected epitopes was analyzed among the various important variants of SARS-CoV-2. Amongst, IYKTPPIKDFGGF was a consensus epitope with no mutation. more details are provided in Table 2.
The designed construct is harboring conserved epitopes of OmpA, OprF, and spike glycoprotein. The alignment of the AbOmpA sequence and OprF 311-341 from P. aeruginosa suggests a great level of identity between the AbOmpA and OprF 311-341 sequence (Fig. 3). Therefore, the identical segment of OmpA (OmpA 301-331 ) was replaced by OprF 311-341 .
Loops (except L3) and internal turns of OmpA β-barrel were replaced with antigenic regions of spike involved in its interaction with ACE2 (RBM), spike cleavage site, and fusion peptide with host cellular membrane. The final design comprised a 348 aa sequence (Fig. 4).

Retrieval of predicted epitopes in construct with appropriate physicochemical properties.
To confirm the efficiency of the construct, the novel sequence was evaluated for antigenicity, flexibility, hydrophilicity, surface accessibility, and beta-turn propensity.
The antigenicity score of the designed construct was 0.7927 while the antigenicity score of S 437-806 was 0.5509. In the new context, the selected epitopes were flexible, hydrophile, surface accessible, and/or a beta-turn (Fig. 5). The epitopes were retrieved by epitope predictors, suggesting an accurate presenting property of the scaffold.   www.nature.com/scientificreports/ AllergenFP predicted the construct as a non-allergen while AlgPred 2.0 hallmarked it as an allergen. The construct was characterized as non-toxic. The theoretical pI and instability index of the construct was calculated as 8.79 and 31.56 respectively. Proteins with an instability index of < 40 are classified as stable.

NSNNLDSKVG
Knowledge-based structural prediction of the construct. Our preliminary approach to finding the most appropriate structure for the construct sequence was to predict the structural classification, topology, and secondary structure of the construct. The perspective obtained from these predictions has paved the way for selecting, quality assessment, and confirmation of tertiary structures.
The designed construct was classified as an OMP. To determine the overall topology of the construct, three topological classification approaches were performed. Although one algorithm out of three did not predict the native OmpA as a ß-barrel outer membrane protein, all approaches have classified the engineered construct as a ß-barrel outer membrane protein with high scores and confidence. This reveals the maintenance of the whole structure and the accuracy of structural replacements. Therefore, the overall integrity of the beta-barrel is expected from engineered OmpA.
The novel antigen is composed of an 8-stranded beta-barrel and a globular C-terminus domain. To get insight into the overall structure of the modified sequence (it is the construct) the topology of the structure was evaluated. PRED-TMBB assigned the designed construct as a beta-barrel outer membrane and scored a value of 2.903. This score was 2.885 for the mature sequence of OmpA. The complete outputs of the beta-barrel predictors are summarized in Supplementary Table S3. As is evident in the Table, the sequence is of a beta-barrel outer membrane protein.  www.nature.com/scientificreports/ Secondary structure. The secondary structure of the designed construct was predicted by two different tools.
The secondary structure components of the designed construct were 11% alpha-helix, 50% beta-strand, and 38% coil. The consistency of the predicted secondary structure with the predicted topology of the construct was visually inspected. The comparison implies the conformity of structural elements with different prediction approaches. This would be a guideline for assessing the quality and accuracy of tertiary structure. Tertiary structures. Overall, 31 3D models were the result of structural prediction of six different approaches.
Along with the consistency of topology and secondary structure components with the predicted structure, the overall quality of the structures was also investigated. The most qualified structure that gained the best scores and showed the best consistency with the predicted topology and secondary structure was selected for further analysis.
One out of five suggested models by GalaxyWEB was assigned as the most qualified structure. Ramachandran plot showed that 90.4% of residues in this model were in the favored region. (A qualified model typically has at least 90% of its residues in the favored region. This model has undergone a refinement process by which 10 refined models were provided (Supplementary Table S4). The epitopes of interest mapped on the 3D structure were surface exposed and accessible (Fig. 6).

Optimized sequence for recombinant expression in E. coli. The protein sequence of the designed
all-in-one antigen was submitted to the codon optimization tool of VectorBuilder to obtain an optimized DNA sequence for E. coli expression. An optimized DNA sequence with GC content of 55.49% and codon adaptation index (CAI) of 0.93 was suggested (Supplementary Fig. S1). Opening energy (mRNA accessibility) of translation initiation sites in the obtained DNA sequence was optimized by TIsigner to be expressed in E. coli. The expression score was predicted 30.3 and 93.24 for the input sequence and the optimized one respectively. The opening energy was calculated as 14.55 and 8.13 kcal/mol for the input sequence and the optimized one respectively.

Discussion
The research community has faced various problems in implementing treatments against SARS-CoV-2, including antigen design, vaccine escape, weak immunity, multiple vaccine shots, and manufacturing costs, to name but a few. A dramatic increase in hospitalization during the COVID-19 pandemic would consequently lead to an upsurge in exposure to nosocomial infections. Among various opportunistic pathogens, A. baumannii and P. www.nature.com/scientificreports/ aeruginosa are the most notorious ones. While these bacterial pathogens are resistant to most available antibiotics, vaccination or passive immunization seem to be the best therapeutic options. The design of a multi-pathogen covering antigen to be used in passive or active immunization could be a proper solution for various challenges raised following recent events. Bacterial outer membrane proteins hold promise for the presentation of foreign peptides (epitopes in the present case) 131 . This approach would require the highly intelligent design of antigens by the implementation of prominent methods such as in silico tools. As is the case in the present study, in silico tools have been used to collect, define structures, and engineer AbOmpA to accommodate and present the foreign epitopes, derived from P. aeruginosa and SARS-CoV-2 antigens. Vaccine design and development require prevailing over some immunological complications. One of the most pronounced concerns is the safety and long-term duration of protection a vaccine can provide 25 . The other challenging complication of vaccination is triggering autoimmune responses 132 . Antibody-dependent enhancement (ADE) is a further concern in which binding between the Fc receptor and the Fc region of IgG is one of the main mechanisms of this phenomenon 133 . In this regard, passive immunization by polyclonal antibodies such as IgY and equine serum raised against protective epitopes could be considered a safer alternative against various viral and bacterial infections 24,134 . As an additional advantage, the passive immunization protocol is less complicated in terms of the required criteria for clinical approval. Vaccination should be performed in healthy individuals while passive immunotherapy could be only administered to hospitalized patients. So, in comparison with vaccination, a smaller population could be affected by the probable side effects of passive immunization.
The first and foremost step in the development of passive and active immunization is the introduction of an appropriate antigen. Rational antigen designs could be harnessed to circumvent these limitations. Viruses can evade the host immune responses using unprecedented mechanisms. Some potential routes are ADE, antigen glycosylation, and altering immune-dominant epitope presentation. ADE is well-characterized in SARS-CoV-2 infections 102,135,136 . Viruses use glycosylation or removal of glycans to escape the host immune system 137 . S glycoprotein of the SARS-CoV-2 is a heavily glycosylated protein, which could induce neutralizing Abs. Therefore, coronaviruses most likely invoke this mechanism to evade host immunity 53 . The introduction of immunodominant non-neutralizing epitopes is another mechanism by which viruses deceive the host immune system. Epitope-based antigen design is an apt strategy to overcome these immune escape mechanisms 137 .
Although in silico tools could predict the potential B-cell epitopes with high accuracy, existing experimental confirmations for these predictions would significantly contribute to the confident selection of the most effective epitopes from the S glycoprotein. For a deterministic selection of neutralizing epitopes, a specific molecular mechanism of virus pathogenesis was taken into account. Thus, peptides involved in spike-ACE2 interaction, spike cleavage, and fusion with the host cell membrane were selected. The selected peptides were partially or completely overlapped with the neutralizing epitopes. The receptor-binding motif (RBM) of the receptor-binding domain (RBD) includes all residues involved in spike-ACE2 interaction. An in silico study revealed that 3 loops and 2 sheets are encompassing all residues involved in this interaction 138 . Therefore, the loops including these residues were selected to target the spike-ACE2 interaction. The fusion peptide of the spike glycoprotein is also known to induce neutralizing antibodies 53 . It contains a glycosylated asparagine residue within its sequence. Expression of this region in prokaryotic hosts would result in non-glycosylated protein. So, the antibodies produced against this recombinant protein could likely not be able to recognize the glycosylated region in the spike of SARS-CoV-2. Moreover, a glycosylated asparagine residue would mask the peptide from immune vision. www.nature.com/scientificreports/ Therefore, to target the fusion peptide without any immunity masking, the "NFSQIL" sequence was removed from the fusion peptide. Due to the selective pressure and error-prone amplification of the coronavirus genome, mutations in its epitopes have become inevitable. Hence, numerous variants have been reported since the early stages of the pandemic and the emergence of new variants is ongoing. A high rate of mutations could potentially result in weakening or even abolishing the immunization effects. To overcome this challenge, our construct harbors five peptides from the spike protein. Among these peptides, at least two peptides are conserved in wild-type and other variants of the spike protein. Thus, it is expected that specific antibodies raised against the designed antigen would provide productivity against various mutants.
QTQTNSPRRARSV epitope contains more than 91% of a motif (i.e. YQTQTNSPRRAR) suggested to be responsible for the super-antigenic activity of SARS-CoV-2. This characteristic could trigger cytokine storms in adults as well as Multisystem Inflammatory Syndrome in Children (MIS-C). Aside from its similarity to neurotoxins and a viral super-antigen, this sequence structurally resembles Staphylococcal Enterotoxin B (SEB) super-antigen 139 . Surprisingly, a monoclonal antibody developed against SEB, 6D3, could bind to PRRA. Thereby, it interferes with the enzymatic cleavage of spike and inhibits in vitro viral entry 140 . QTQTNSPRRARSV also harbors a peptide that is identical to the human proteome. This peptide is suggested to be involved in the induction of autoimmune responses 132 . Therefore, this epitope should be removed from antigens designed as vaccine candidates; however, antibodies developed against this epitope could act as a Swiss army knife for passive immunization against SARS-CoV-2. In addition to the neutralization of SARS-CoV-2 and prevention of its super-antigenic activity, these antibodies could inhibit autoantibody triggering by epitope masking 141 .
It has been suggested that the production of high-potency neutralizing antibodies against RBM is affected by the weak presentation of MHC-II binders in this region of SARS-CoV-2 spike glycoprotein 142 . In this regard, the incorporation of linear B-cell epitopes within a potent antigen harboring strong MHC-II binders could assure robust induction of antibodies against the B-cell epitopes of interest.
OmpA and OprF are highly conserved antigens of A. baumannii and P. aeruginosa, respectively 21,143 . The inclusion of experimentally validated B-cell epitopes from these two orthologous OMPs could assure the elicitation of protective antibodies against A. baumannii and P. aeruginosa. OmpA is one of the most promising antigens of A. baumannii, which triggers high titers of protective antibodies 21,22,37 . It had been demonstrated that recombinant OmpA purified in denaturing conditions could also raise protective antibodies against A. baumannii. This property was attributed to linear B-cell epitopes of OmpA 22,37 . All epitopes of the designed construct were validated linear B-cell epitopes. The similarity of these epitopes with human peptides is less deleterious in passive immunization. It has been demonstrated that epitope masking with passively administered epitope-specific IgG could suppress IgG production against the given antigen 141,144 . Hence, passive immunization by specific antibodies raised against the all-in-one antigen could suppress autoimmune antibody responses against epitopes shared with human peptides. Hence, these similarities could be considered an advantage.
Antibodies raised against the linear B-cell epitopes in the new context (i.e. the designed construct), could recognize these epitopes in the original antigens (OmpA, OprF, and spike). Hence, the all-in-one antigen could be purified in denaturing conditions. Several multi-epitope antigens have already been designed against SARS-CoV-2. Predicted B and T cell epitopes of these multi-epitope antigens were fused by various repeats of known peptide linkers [68][69][70][71][72] . It has been demonstrated that increasing the copy number of a given sequence could enhance the specific antibody responses [145][146][147] . Therefore, increasing the repeats of spacers (linkers) is deleterious to misspend antibody responses. Several recombinant antigens, harboring epitope 8 (OprF 311-341 ) as a protective region, are in various phases of clinical trial studies (reviewed in 23 ). The OprF 311-341 epitope shares identity with an immunodominant epitope of OmpA in P. aeruginosa. OmpA has a nuclear localization signal (NLS) at the C-terminal domain, which confers the cytotoxicity for this antigen. K 320 and K 322 are located at the NLS and substitution of these residues by Alanine could decrease OmpA cytotoxicity 148 . Interestingly, these substitutions have naturally occurred in the OprF 311-341 epitope. Thus, replacing a similar region in OmpA with the OprF 311-341 epitope in the designed construct decreases the antigen toxicity and enables its high dose administration. Among 4 validated epitopes of OmpA retained in the designed construct, only one epitope underwent a minor change by this replacement.
Recently, a novel OmpA-derived antigen has been designed in which the last 15 residues of CTD-OmpA were removed from the designed antigen. This region was undesirable for its antigenicity and B-cell epitope properties (hydrophilicity and flexibility). Moreover, K 320 and K 322 located at the NLS were substituted by Alanine, and loop 4 (NADEEFWN) of the 8-stranded barrel (in the two-domain conformer of OmpA) was replaced by loop 3 (YKYDFDGVNRGT RGT SEEGTL) 66 . It has been suggested that the designed antigen is a more antigenic and less toxic immunogen. In the current design, residues of loop 4 were replaced by a spike epitope. The last 15 residues of CTD-OmpA were removed.
High epitope density could significantly enhance the antigenicity and immunogenicity of the antigen of interest [145][146][147]149 . This criterion was met by the number of encompassed epitopes, the number of repeats for a given epitope, and the spatial and conformational density of epitopes. The designed all-in-one construct contains five peptides of spike glycoprotein condensed at the N-terminal domain (NTD) of the antigen (consisting of about 200 residues). Transmembrane β-strands of the OmpA barrel could act as natural spacers for the embedded peptides of S glycoprotein in the designed construct. The structural analyses revealed that the NTD is a β-barrel presenting the epitopes exposed at both sides of the barrel in the native structure. So, the epitopes of interest are more accessible compared to tandemly fused multi-epitope vaccines. One peptide was repeated at the periplasmic side of the barrel to increase its number and spatial density. Compared with multi-epitope antigens fused sequentially, it would be expected that the native structure of epitopes (at least those for OmpA and OprF) retained in the designed all-in-one construct. www.nature.com/scientificreports/ Although based on in silico analyses, it would be expected that the designed all-in-one antigen could trigger robust protective and neutralizing antibodies against P. aeruginosa, A. baumannii, and SARS-CoV-2, experimental confirmations should be carried out in future studies. Safety considerations such as allergenicity, toxicity, autoimmune responses, and ADE should be assessed in addition to efficacy, in further pre-clinical and clinical studies.

Conclusion
In silico approaches provide appropriate tools for rational design, evaluation, and engineering of a molecule that harbors immunogenic regions of target genomes. These tools are imperative for the design of multi-epitope constructs that could be useful in active and passive immunizations. OmpA from A. baumannii is a proper scaffold for the design of multi-epitope constructs. In the present study, OmpA was engineered to present foreign epitopes embedded within its sequence. The risk of facing nosocomial pathogens in the hospital environments, the economical and executive burdens of manufacturing multiple antigens, and the inevitability of multiple booster shot administration have made the idea of multi-target antigens an appealing one. www.nature.com/scientificreports/